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Mol Carcinog. Author manuscript; available in PMC Nov 1, 2006.
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Estrogen Receptor α Increases Basal and Cigarette Smoke Extract-Induced Expression of CYP1A1 and CYP1B1, but not GSTP1, in Normal Human Bronchial Epithelial Cells

Abstract

Gender-specific estrogen receptor α (ERα) expression may plausibly influence lung carcinogenesis in females. Initial genome-wide microarray studies confirmed that carcinogen metabolism genes (CYP1A1, CYP1B1) were those most responsive to cigarette smoke extract (CSE) in normal bronchial epithelial (NHBE) cells. These two genes encoding phase I bioactivating enzymes and the GSTP1 gene encoding a phase II deactivating enzyme were then tested for induction by ERα. NHBE cells (native ERα) were transfected with wild-type ERα-adenoviral constructs, and then exposed to CSE, 17β-estradiol (E2), and/or the ERα inhibitor, ICI 182,780. The expression levels of CYP1A1, CYP1B1 and GSTP1 were then determined by RNA-specific quantitative RT-PCR and immunoassay. ERα increased the basal expression of CYP1B1 4.04-fold (p<0.01) at the mRNA level and 6.5-fold at the protein level. ERα also increased the CSE-induced mRNA expression of CYP1B1 2.26-fold (p<0.01), but not the protein expression. ERα did not alter the CYP1A1 mRNA levels, but did increase protein expression 2.0-fold (p<0.01) on CSE exposure, and 6.2-fold (p<0.01) upon E2 exposure. These effects could be inhibited by ICI 182,780. ERα did not alter the expression of GSTP1. ChIP assay confirmed ERα binding to CYP1B1 promoter near the transcription start site. These results suggest that ERα regulates the CYP1B1 expression at a transcriptional level, and CYP1A1 expression at a translational level. These data raise the possibility that inter-gender differences in expression of ERα that are known to exist in human lung may contribute to inter-individual expression differences in CYP1A1 and CYP1B1, and to differences in carcinogen metabolism and mutation.

Keywords: estrogen receptor, CYP1A1, CYP1B1, GSTP1, cigarette smoke extract
Abbreviations: CYP1A1/1B1, cytochrome P450 1A1/1B1; GSTP1, glutathione S-transferase pi; CSE, cigarette smoke extract; NHBE, normal bronchial epithelial cell; ChIP, Chromatin Immunoprecipitation; ERα, estrogen receptor α; AD-CON: adenovirus control vector; AD-ER: estrogen receptor α recombinant adenovirus expression vector; DMSO: dimethyl sulfoxide; XRE, xenobiotic response element; ERE, estrogen response element; AhR, aromatic hydrocarbon receptor; ARNT, aromatic receptor nuclear translocator; PAH, polycyclic aromatic hydrocarbon; E2, 17β-estradiol

INTRODUCTION

There is a significant literature suggesting a possible enhancement of female susceptibility to lung cancer (18), although recent studies with contrary epidemiological findings have been reported (9, 10). Higher levels of polycyclic aromatic hydrocarbon (PAH) metabolite-DNA adducts occur in woman when smokers are divided into quartile groups according to adducts per pack year (11). Although dosimetric factors more precise than simple pack-years have not been thoroughly studied as they relate to female lung cancer risk, there remains support in the epidemiologic literature for the position that woman are at higher risk for lung cancer, and convincing evidence that they have different prognoses once diagnosed (1214), at any given level of smoking.

Estrogen receptors α and β (ERα, ERβ) are ligand-binding transcription factors, and members of the nuclear receptor family (15, 16). ERs bind estrogen response elements (EREs) encoded in genomic DNA, as well as other DNA binding proteins, and they classically activate transcription in an estrogen-dependent manner. The patterns of expression of two ER forms in human lung tissue has been described (17): ERα is expressed more often in the lungs of women than men, while ERβ is expressed with approximately equal frequencies in the lungs of men and women. Recent studies suggest that ERs can directly interact with the aromatic hydrocarbon receptor (AhR) and aromatic receptor nuclear translocator (ARNT) (18, 19). The AhR/ARNT complex is also a ligand-dependent transcription factor; PAH-activated AhR heterodimerizes with ARNT and activates the transcription of carcinogen– and estrogen– metabolism target genes, such as CYP1A1 and CYP1B1, through xenobiotic response elements (XREs) (20, 21). This literature implies that ERs plausibly affect the regulation of carcinogen metabolism gene expression.

Because tobacco-induced expression of phase I bioactivating and phase II deactivating carcinogen metabolism enzymes in human lung has been suggested in observational studies to demonstrate wide inter-individual and inter-gender variation (22, 23), as well as putative corollary changes in mutagenesis, we have explored the possibility that qualitative differences in expression of ERα underlie some of this inter-individual variability in quantitative carcinogen– metabolism gene expression. In the current study, we experimentally replicate the gene–environment interaction present in normal human bronchial epithelial (NHBE) cells exposed to tobacco smoke during early initiation of lung carcinogenesis. We confirm, by genome-wide expression microarray scan, that the most CSE-responsive genes are members of the CYP1 family, and we demonstrate the impact of experimental ERα co-expression on the CSE-induced expression of CYP1A1, CYP1B1, and GSTP1 native transcripts in NHBE cells. We further explore potential mechanisms by which ERα enhances CYP1B1 expression, using reporter constructs and chromatin immunoprecipitation assay (ChIP).

MATERIALS AND METHODS

Microarray scan for CSE responsive genes

Total RNA was prepared from NHBE cells (RNeasy®, Qiagen, Valencia,CA) that had been exposed to 1.0 μg/ml CSE and DMSO as control for 18 h. After gel and capillary electrophoresis (Agilent Bioanalyzer® 2100, Palo Alto, CA) confirmation of total RNA integrity from two separate experimental NHBE exposures, biotinylated cRNA was generated using a MessageAmp™ aRNA Kit (Ambion, Austin, TX) with minor modifications to the manufacturer’s instructions. This cRNA was hybridized to Human U133 Plus 2.0 GeneChip arrays (Affymetrix, Santa Clara, CA) in replicate samples, in the Wadsworth Center Microarray Core Facility. Pooled data were checked against results of individual runs for reproducibility, and were analyzed using GeneTraffic® (Iobion, La Jolla, CA) software.

Constructs

Recombinant adenovirus constructs: The human ERα construct with a mutation at Gly400Val (24) was reverted to wild type by site-directed mutagenesis, whereby oligonucleotide-directed PCR mutagenesis (QuikChange® multi site-directed mutagenesis kit, Stratagene, La Jolla, CA) generated a wild-type ER construct, using the synthetic oligonucleotide: 5’-CCATGGAGCACCCAGGGAAGCTACTGTTTGC-3’( the changed nucleotide is underlined). The high-fidelity PCR was performed by PfuTurbo DNA polymerase (Stratagene) to introduce KpnI and SalI sites into the 5’and 3’ ends of the ER sequence, using forward primer: 5’-TACTATGGTACCCGGCCACGGACCATG-3’ and reverse primer: 5’-TACTATGTCGACGCCAGGGAGCTCTCAG-3’(the underlined portions are introduced restriction sites). PCR product was purified from an agarose gel, digested with KpnI and SalI, and inserted into the KpnI/SalI site of pAdTrack-CMV vector (Stratagene) to generate an AdTrack-ER expression construct. The desired base changes, inducing reversion of the coding sequence to that of wild-type ERα, were verified by direct cycle sequencing of the entire ER insert. Recombinant adenoviruses were produced by AdEasy XL Adenoviral vector system (Stratagene). Each construct contained a green fluorescent protein (GFP) coding sequence downstream from the CMV promoter, for tracking of transfectionion efficiency. Briefly, AdTrack-ER was linearized with PmeI and was transformed into BJ5183-AD-1 cells. The recombinant adenovirus ER construct (R-ER) was identified by PacI digestion. Similarly, a recombinant adenovirus control construct (R-CON) was created by transforming linearized AdTrack-CMV vector into BJ5183-AD-1 cells. Recombinant adenoviruses, AD-ER and AD-CON, were produced by transfection of PacI digested R-ER or R-CON into AD-293 cells. The wild type ER sequence insert was further confirmed by PCR and sequencing. CYP1B1 promoter luciferase reporter construct was described previously (25). Estrogen response element (ERE) Luciferase reporter construct and Xenobiotic response element (XRE) Luciferase reporter construct were generous gifts of Drs. E. Bessette and L. Kaminsky (26,27).

Cell culture

NHBE cells were primary cultures obtained from a commercial source (BioWhittaker, Inc., Walkersville, MD). ERα–negative background was confirmed by RT-PCR and Western blot (data not shown). NHBE cells were maintained in BEGM (Bronchial Epithelial Cell Growth Medium) medium (BioWhittaker, Inc.) containing BEBM (Bronchial Epithelial Cell Basal Medium) supplemented with 52 μg/ml bovine pituitary extract, 0.5 μg/ml hydrocortisone, 0.5 ng/ml human epidermal growth factor, 0.5 ng/ml epinephrine, 10 μg/ml transferrin, 5 μg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyrinine, 50 μg/ml gentamicin, and 50 ng/ml amphotericin-B, at 37 °C in a humidified 5% CO2 atmosphere. All experiments were performed on cells from a single female donor, lot, aliquot and passage.

Adenoviral transfection

Adenoviral transfectionions were performed in triplicate in 24-well or 6-well plates. Each well was plated with 1 × 105 cells (for 24-well plate) or 5 × 105 cells (for 6-well plate) on passage 4, for 24 h before transfection. The virus of AD-ER or AD-CON, 0.02 ml/cm2 of 1:320 dilution normalized to obtain approximately 90% transfection efficiency (as tracked by GFP fluorescent microscopy) was incubated with NHBE cells for 2 h, and the media was then replaced with growth medium (BEGM). These ER- transfectants were ready for use after 24-h incubation.

Reporter construct transfection

For ERE-Luc, XRE-Luc and p1B1-Luc luciferase reporter construct studies, transfection was performed on NHBE transfected with AD-ER or AD-CON for 24 h in 24-well plates using 0.2 μg of each plasmid, together with 0.2 μg of reference PRL-TK vector (Promega, Madison, WI) expressing renilla luciferase constitutively; the latter serves as an internal control of reporter construct transfection efficiency. DNA was combined with 1 μl of LipofectAmine 2000 in 50 μl of Opti-MEM reduced serum medium (Invitrogen, Carlsbad, CA).

In vitro exposure

For quantative mRNA transcript studies of NHBE, carried out 24 h after the initial AD-ER transfectionion, the medium of NHBE was replaced with growth medium (BEGM) containing CSE (1.0 μg/ml media ), 1.0 ng/ml media 17β-estradiol, and/or 0.1μM of the ERα inhibitor ICI 182,780, dissolved in 100% dimethyl sulfoxide (DMSO). Cells were harvested after an additional 3, 12, or 18 h of exposure, for total RNA extraction. Control cells were exposed to 0.1% (v/v) DMSO vehicle only. For protein studies, cells were harvested after an additional 24 h of exposure, which was chosen on the basis of optimum GFP expression. CSE was purchased from Murty Pharmaceutical Inc. (Lexington, KY). It was prepared using a Phipps-Bird 20-channel smoking machine designed for Federal Trade Commission testing. Kentucky standard cigarettes 1R3F (University of Kentucky, KY) were smoked, and the particulate matter was trapped onto filters. The amount of CSE obtained was determined by weight increase of the filter. CSE was dissolved from the filter in dimethyl sulfoxide by sonication to yield 4% solution and stored at −20 °C. The average yield of CSE was 21 mg per cigarette including 1.3 mg nicotine.

For reporter construct studies, after initial transfection incubation for 5 h, the medium was replaced with growth medium (BEGM) containing CSE (Murty Pharmaceuticals, Inc.), 17β-estradiol, and/or the ERα inhibitor ICI 182,780, dissolved in DMSO as above, and cells were harvested after an additional 24 h (the previously determined maximal–effect time point). In each condition, the final concentration of DMSO was adjusted to 0.1% (v/v). The cells were lysed by Passive Lysis Buffer (Promega). The activities of firefly and renilla luciferases were determined by Dual-Luciferase reporter assay system (Promega). Firefly luciferase activity was then numerically normalized to renilla luciferase activity.

RNA-specific Universal Reverse Transcription and PCR

Total RNA was prepared by RNeasy Mini Kit (Qiagen). RT was performed by universal RT primer as previously described (28, 29), avoiding genomic DNA–encoded false positives in the RT-PCR that are yielded by pseudogene-encoded sequences (e.g., GSTP1 and 36B4).

RT was performed with 2.0 μg of total RNA, using Superscript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD) as follows. RNA template was added to 1 μl of RT primer (100μM universal RT primer (URT); Table 1), 1 μl of dNTP mix (each base, 10 mM) and DNase/RNase-free water, to a volume of 13 μl. The solution was incubated at 65 °C for 5 min and then cooled to 4 °C. A master mix containing 4 μl of 5x first-strand buffer (Gibco/BRL Life Technologies, Gaithersburg, MD) and 2 μl of 0.1 mM DTT per RT sample was prepared, and was added to each sample. The sample was then incubated at 42 °C for 2 min. SuperScript II Reverse Transcriptase (Gibco/BRL Life Technologies) was added (1 μl), and the samples were incubated at 42 °C for 50 min, followed by a further incubation at 70 °C for 15 min.

Table 1
Primers for quantitative RT-PCR.

Quantitative PCR was then performed in the LightCycler® (Roche, Indianapolis, IN) thermocycler using CYP1A1, CYP1B1, GSTP1, PgR , HEM45, 36B4 and 28S RNA-specific primers (Table 1). RT-PCR target transcript results were normalized to expression levels of the internal reference housekeeping gene 36B4 which is relatively unaffected by hormonal factors (30), or to 28S ribosomal RNA.

Western immunoblotting

Microsomes for the analysis of CYP1A1 and CYP1B1 proteins were prepared as previously described (31). For analysis of ERα and GSTP1, cultured cells were lysed in SDS sample buffer described previously (32). These microsomal and cell-lysate samples were subjected to electrophoresis in 10% acrylamide NuPAGE Novex Bis-Tris denaturing gels (Invitrogen) as recommended by the manufacturer. Proteins were blotted onto 0.2um Nitrocellulose membranes (Invitrogen), and blots were blocked in Blotto B (1% bovine serum albumin, 1% non-fat dry milk, 0.05% Tween20 in PBS) for 1 h at room temperature. Blots were then probed with goat polyclonal anti-rat CYP1A1/2 antibody (Daiichi, Tokyo, Japan) diluted 1:1000 in Blotto B; ERα-specific antibody HC-20 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:100; rabbit polyclonal anti-human CYP1B1 antibody (33) diluted 1:400; or rabbit polyclonal anti-human GSTP1 antibody (Oxford Biomedical Research,Inc.,MI) diluted 1:500 in blocking buffer. Blots were then probed with HRP-conjugated anti-rabbit IgG or anti-goat IgG (Sigma), at 1:10,000 dilution. Immunoreactive proteins were detected by using the ECL western blotting reagent (Amersham Biosciences, Piscataway, NJ). Proteins were detected by enhanced chemiluminescence on Kodak X-OMAT film (Sigma-Aldrich) and quantified by densitometry with a Molecular Dynamics scanning laser densitometer.

Chromatin Immunoprecipitation (ChIP) Assay

NHBE cells were transduced with AD-ER or AD-CON as described above , then exposure to 1nM E2 and 1ug/ml CSE for 18 hours and ChIP assay was carried out essentially using the method of Braunstein and coworkers (34) briefly, treated NHBE Cells were cross-linked by addition of 1% fformaldehyde to the medium for 10 min. Crude nuclei prepared by hypotonic lysis were resuspended in 100 μl SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), sonicated under conditions that reduced DNA length to between 200 and 1000 base pairs, and debris removed by centrifugation. The chromatin solution was diluted 10-fold in IP buffer (34) and precleared for 45′ at 22–25 °C on protein A beads (Pierce) preadsorbed with sonicated single-stranded DNA. The chromatin solution was then incubated with anti-ERα antibody (1μl; Santa Cruz) for 45 min at 22–25 °C, and immune complexes collected with protein A beads preadsorbed with sonicated single-stranded DNA. Following washes and elution (34), cross-links were reversed by heating at 65 °C for 4–5 hr; DNA was recovered by phenol extraction and ethanol precipitation. Specific sequences in the immunoprecipitates were detected by PCR. The forward and reverse primers for region encompassing the putative ERE sequence (35) were 5'- TGGAGGTGGCTGTGATGA-3' and 5'- CACAACTGGAGTCGCAGAA -3', respectively. PCR analyses were performed using the Advantage-GC Genomic PCR kit (Clontech, Palo Alto, CA) as follows: after an initial denaturation at 95 °C for 1 min, denaturation at 95 °C for 10 s, touchdown annealing at 62 °C, 30s for 5 cycles, 58 °C, 30s for 35 cycles, and extension at 72 °C for 30s, for a total of 40 cycles. A final extension step at 72 °C for 7 min terminated the reaction. The PCR product was electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining.

Statistical analyses

For the microarray studies, normalization and statistical analysis were performed with GeneTraffic UNO v2.8 software (Iobion). The set of four arrays was normalized using the Robust Multichip Analysis algorithm (RMA) (36), and probe sets were excluded from further analysis if the normalized intensity value did not reach 200 in at least two (50%) of the hybridizations. Unpaired t-tests were performed on the RMA–transformed data, utilizing the variance stabilization and Benjamini-Hochberg multiple testing (37) correction options. For quantitative RT-PCR studies, triplicate samples under various conditions were compared by t-test, using MS-Excel software. Figure error bars depict standard deviations, and differences were considered significant if p<0.05.

RESULTS

Genome–wide scan for CSE induction

Initial genome–wide expression studies employing the Affymetrix human U133 plus 2.0 GeneChip suggested that five genes were significantly induced > 2–fold (Table 2) on NHBE exposure to cigarette smoke extract for 18 h. CYP1A1 and CYP1B1 were the most highly CSE–induced genes among 36,000 genes included in this GeneChip. All known cytochrome P450, glutathione S-transferase, sulfotransferase and UDP glycosyltransferase family enzymes are included on this latest version GeneChip. The results of quantitative real-time RT-PCR (data not shown) confirmed this CSE induction; vector-only transfected NHBE cells(ER-) were increased 4.45 fold for CYP1A1 and 7.94 fold for CYP1B1 by 18 h of CSE exposure at 1.0 μg/ml. GSTP1 expression was unchanged by CSE exposure.

Table 2
Genes significantly induced over two fold by CSE-induction using expression microarray.

Expression and function of ERα

NHBE cells were transfected with AD-ER or AD-CON. The expression of ERα was demonstrated by Western immunoblotting (Figure 1). We chose a 1:320 dilution in this study, because at that titer, the transfection efficiency was about 90% by green fluorescent protein (GFP) integrated in the adenoviral expression constructs, in preliminary titration studies. The expressed ERα drove a positive control ERE reporter construct expression in an estradiol dose–dependent manner (Figure 2). As additional positive controls, the expressed ERα enhanced the expression of the known, ER-regulated PgR 7.47–fold and that of HEM45 9.04–fold (Figure 3). These results collectively demonstrate that transfected ERα was expressed and was functional.

Fig. 1
Detection of ERα in AD-ER and AD-CON transfected NHBE cells by Western immunoblotting. Two titers of the recombinant adenovirus (1:160 and 1:320) were used to transfect NHBE cell. Whole cell lysate was prepared 24 hours after transfection. 20μg ...
Fig. 2
The effect of expressed ERα on ERE-luciferase reporter construct (ERE-Luc) expression. NHBE cells were transfected with AD-CON or AD-ER adnovirus for 24 hours, then transfected ERE-luciferase construct and exposure to 0.1% DMSO, 17β-estradiol ...
Fig. 3
Effect of expressed ERα on the expression of PgR and HEM45. NHBE cells were transfected with AD-CON or AD-ER adnovirus and then exposed to 0.1% DMSO, 1nM 17β-estradiol (E2) for 3 hours. The mRNA levels of PgR (a), HEM45 (b) were determined ...

Effect of AD-ER transfectionion on CYP1A1, CYP1B1 , GSTP1 mRNA Expression

NHBE cells were transfected with AD-CON or AD-ER for 24 h, and were then exposed to combinations of DMSO, CSE, 17β-estradiol (E2) and ICI 172,780. mRNA levels of CYP1A1, CYP1B1 ,GSTP1 and 36B4 after 0, 3, 12 and 18-hour exposure. Maximal expression was observed at 3-hour exposure. CSE could induce mRNA expression of CYP1A1 and CYP1B1 4.3 to 6.8-fold in both AD-ER and AD-CON transfected cells, but not that of GSTP1. This result is consistent with the gene expression microarray (Figure 4). Compared to ER- cells, ERα significantly increased CYP1B1 basal expression 4.04–fold (p<0.01) with medium alone, and CSE-induced expression 2.26–fold (p<0.01) (Figure 4a). Estradiol at 1nM affected neither the basal nor the CSE-induced CYP1B1 expression, in either ER– or ER+ cells (Figure 4a). The enhancer effect of ERα was inhibited by 0.1 μM ICI 182,780 (Figure 4a). ERα did not alter the mRNA expression of CYP1A1 and GSTP1 (Figure 4b, 4c). Similar results were observed at 12 and 18-hour exposure (data not shown). We also checked the expression of CYP1B1 at lower transfectionion titer (1:640), where transfection efficiency was 80% (versus 90% with 1:320) and results were predictably consistent with those reported at 1:320 titer, but of lower magnitude, due to lower transfection efficiency (Figure 4d)

Fig.4
Effect of expressed ERα on the expression of CYP1A1, CYP1B1 and GSTP1. NHBE cells were transfected with AD-CON or AD-ER adnovirus for 24 hours and then exposed to 0.1% (v/v) DMSO as vehicle control; 1nM 17β-estradiol (E2); 1.0μg/ml ...

Effect of AD-ER transfectionion on CYP1A1, CYP1B1 , GSTP1 protein Expression

CYP1A1, CYP1B1 , GSTP1 protein was detected by Western immunoblot in the treated NHBE cells as described above. Results showed that CSE induced CYP1A1 and CYP1B1 protein expression in both AD-ER and AD-CON transfected cells, but not GSTP1 expression (Figure 5). Compared to ER- cells, ERα increased the basal expression of CYP1B1 by 6.5-fold (p<0.01; Figure 5a), but not the CSE-induced CYP1B1 protein level. ERα also increased CYP1A1 protein expression by 2.0-fold (p<0.01) upon CSE exposure, and by 6.2-fold upon E2 exposure (p<0.01; Figure 5b). GSTP1 protein expression was not affected by either CSE or by ERα status (Figure 5c).

Figure 5Figure 5Figure 5
Effect of expressed ERα on the protein expression of CYP1A1, CYP1B1 and GSTP1 in NHBE cells. NHBE cells were transfected with AD-ER and AD-CON for 24 hours, then exposure to 0.1% DMSO, 1nM 17β-estradiol (E2); 1.0μg/ml CSE; 1.0μg/ml ...

Binding of ERα to CYP1B1 promoter

A putative ERE sequence in the human CYP1B1 gene between –63 and –49 (Figure 6A) has recently been reported (35). The sequence (AGGTCGCGCTGCCCT) is different from the consensus ERE (AGGTCANNNTGACCT, from the Xenopus vitellogenin A2 gene) (38) in two bases. To confirm that ERα is associated with the putative ERE on the human CYP1B1 gene in NHBE cells, chromatin immunoprecipitation (ChIP) assays were performed. DNA was extracted from NHBE cells transfected with AD-ER or AD-CON and exposure to 1nM E2 and 1ug/ml CSE for 18 h after cross-linking treatment and incubated with specific anti-ERα antibodies. PCR was performed with a primer set for the region (–235 to +46), which includes the putative ERE. In AD-ER transduced NHBE cells, immunoprecipitants obtained with anti-ERα antibodies generated a distinct PCR product with the primer set, whereas in AD-CON transduced NHBE cells, no distinct band was observed (Figure 6B). These results indicated that ERα binds to a region containing the putative ERE, and suggest that ERα directly regulates the CYP1B1 promoter activity.

Figure 6
Chromatin immunoprecipitation (ChIP) assay of promoter region of CYP1B1 gene. (A): the amplified promoter region of CYP1B1. The underlined is the putative estrogen response element (ERE) binding site. (B): NHBE cells were transfected with AD-ER or AD-CON ...

DISCUSSION

We have shown by genome-wide expression microarray scan, that CYP1B1 and CYP1A1 are the two genes that are most highly responsive to tobacco smoke in normal primary isolates of bronchial epithelial cells. We then proceeded to confirm ERα regulation of these two phase I genes, but not the phase II GSTP1 genes, in several relevant exposure contexts, using RNA-specific quantitative RT-PCR, and Western immunoblots. Finally, we detected a putative corresponding estrogen response element (ERE) in the CYP1B1 promoter.

For the medium-only control, ERα increased basal expression of CYP1B1 for both mRNA and protein by 4.04- and 6.50-fold, respectively. The ERα-induced increment for DMSO vehicle controls was less, at 2.26-fold. Both conditions suggest that ERα effects on this pathway can be qualitatively independent of ligand. For mRNA, the reason for the difference between the ERα-induced increments in medium only versus DMSO vehicle controls is a matter of speculation, with no direct studies available. However, DMSO at 4% is known to release ribosomes from mRNA transcripts (39), albeit the relevance to the lower DMSO concentrations employed in this study (0.1%) is unclear. Whether a similar release phenomenon occurs with transcription factor binding as that for observed for ribosomes is unknown.

In CSE-induced expression of CYP1B1, ERα increased mRNA expression only, but not protein. It could be speculated that this indicates saturation of protein translation, as CSE induced CYP1B1 expression by 31-fold on protein level. The saturated expression was also observed in expression of CYP1B1 promoter luciferase reporter constructs when CSE concentration reached 10μg/ml (data not shown). Further studies are warranted to clarify the kinetics of transcriptional and translational regulation of this gene.

ChIP assays demonstrated that ERα could bind to the putative ERE sequence. This confirms a mechanism by which ERα likely affects CYP1B1 expression at the transcriptional level. Additionally, it has been reported that some estrogen-regulated genes are indirectly regulated by the cooperation of Sp1 and ER within a GC-box and ERE half site (40, 41), In the human CYP1B1 gene promoter, two Sp1 binding sequences have been reported to be located at −84 to −89 and −68 to −73, (42) that are near the putative ERE (−49 to −63). It will be important, in future studies, to clarify whether Sp1 and ER cooperatively regulate the transcription of the CYP1B1 gene.

ERα did not alter the CYP1A1 mRNA levels, but did increase protein expression 6.2-fold (p<0.01) upon E2 exposure and 2.0-fold (p<0.01) on CSE exposure. This result suggests that ERα regulates CYP1A1 expression at the translational level. While the mechanism is still unclear, it is known that the effects of ERα status on the expression of CYP1A1 and CYP1B1 differ in a tissue-specific manner (43~46). ERα as a transcriptional factor is well known, but as a translational factor is not defined. Therefore, it will be of some significance to explore how ERα regulates CYP1A1 expression at this translational level.

There is a large literature confirming the critical role of the two phase I enzymes encoded by these genes both for PAH and 17β-estradiol bioactivation. It has been shown that CYP1B1 is the more abundant species in human lung (47); the phase II enzyme expressed in highest abundance in human lung is GSTP1 (22, 48). This study confirms that ERα has a potential role in significantly enhancing the expression of key bioactivating genes (i.e., CYP1A1, CYP1B1) in the carcinogen metabolism pathway. The implication is that phase I (CYP1A1, CYP1B1) metabolism enhancement, in the absence of similar, coordinate enhancement of the phase II deactivation gene (GSTP1), may serve as a mechanism for enhanced female gender-related susceptibility to tobacco- or estrogen-mediated mutations.

Classically, ERα binds to estrogen response elements (EREs) and activates transcription of estrogen target genes in an estrogen-dependent manner (49~53). Given recent confirmation of Ahrligand mediation of ER-mediated estrogen signaling by direct ER-AhR(18) or ER-Arnt (19) interaction, results from the current study reinforce the data from our prior studies that those individuals who express ER in lung (17) demonstrate enhanced expression of CYP1B1 (22).

To demonstrate if ER-AhR cross-talk was also involved in the ERα enhanced CYP1B1 expression, XRE luciferase reporter assay and CYP1B1 promoter luciferase assay were performed (data not shown). These luciferase reporter assay results did not demonstrate the ERα enhancement effect seen in the native CYP1B1expression, indicating that ER-AhR cross-talk are not apparently involved in this ERα mediated enhancement of CYP1B1 expression.

A putative ERE motif (–63 and –49) in the CYP1B1 promoter has been recently confirmed (35), and can up-regulate the CYP1B1 reporter construct expression mediated by ERα. Our chromosome immunoprecipitation (ChIP) assay did confirm the ERα binding to this putative ERE sequence in CYP1B1 promoter. However, ERα did not increase the CYP1B1 promoter (containing 1.5kb of native sequence, and the putative ERE) activity, as measured in a luciferase reporter construct expression (data not shown). This indicates that the elements in other regions of CYP1B1 promoter may be cooperatively involved in ERα regulation of CYP1B1. There are three reported components in the promoter (42): essential elements located between nt-164 and nt+25; enhancer elements are located between nt 1500 and nt 1356 and between nt 1022 and 835; negative regulatory elements are located between nt 1356 and 1022 and between nt 835 and 164. Increasing the size of the promoter sequence analyzed may aide the identification of the essential promoter structure for ERα regulation, even though the ERα binding site demonstrated by ChIP was within the 1.5kb fragment studied. Alternatively, epigenetic or other regulatory features that are not well-represented in reporter constructs may be at play.

A strength of the current study is the use of non-malignant, non-transformed primary lung epithelial cells that are likely to reflect, with reasonable accuracy, the cellular environment for the earliest steps in carcinogen bioactivation in the native epithelium of the human target organ being studied, as compared to already-transformed or malignant cells. Corollary attempts were made to employ relevant concentrations of estradiol (0.1 nM, equivalent to post-menopausal, and 1.0 nM, equivalent to premenopausal plasma levels) and of CSE (0.1 and 1.0 μg/ml; only estimates of bronchial and alveolar lining fluid concentrations currently exist) that are reasonably representive of human in vivo exposure conditions. ERα was over-expressed in NHBE cells (Figure 1), compared with the native level in MCF7 cells, when 90–95% of NHBE cells were transduced. This overexpression was due to strong CMV promoter directly upstream of the ERα coding sequence, and not due to over-transfectionion (data not shown).

In summary, we have demonstrated ERα enhancement of expression of two phase I carcinogen and estradiol bioactivating enzymes, without corollary enhancement of the lung’s main phase II deactivating enzyme. This could be hypothesized to confer female gender related susceptibility to carcinogen- or estrogen-mediated malignancies. Mechanisms at play for this ERα mediated up-regulation may include direct promoter region binding. These data additionally suggest that sequence or structure that is further 5’ upstream, epigenetic structure, higher order protein-protein interactions, or translational regulation may separately, or in aggregate, play important roles in ERα up-regulated CYP1B1 and CYP1A1 expression, and therefore in individual carcinogen bioactivating pathway phenotypes.

Acknowledgments

We thank Guoyu Ling, PhD for aid with adenovirus transfection, Erin Bessette, PhD and Laurence Kaminsky, PhD for the XRE reporter construct; and the Wadsworth Center Genomics, Molecular Genetics, and Biochemistry Cores. Supported by ALA-NENYS (Research Fellowship, to WH); NIH-R21 CA 94714 (to SDS); NIH-R01 CA 10618 (to SDS).

Footnotes

Supported by: Supported by ALA-NENYS (Research Fellowship: RT-84-N to WH); NIH-R21 CA 94714 (to SDS); NIH-R01 CA 10618 (to SDS).

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